Metabolic Dysfunction-Associated Fatty Liver Disease on Distinct Microbial Communities at the Bacterial Phylum Level.

INTRODUCTION: Limited data are available on the correlation between microbial communities and metabolic dysfunction-associated fatty liver disease (MAFLD). This study aimed to evaluate the influence of MAFLD on diverse microbial communities. METHODS: We recruited 43 patients with a nonviral liver disease. Enrolled patients were divided into two groups according to MAFLD criteria. The fecal microbial composition was evaluated using the variable V3-V4 region of the 16S ribosomal RNA region, which was amplified using polymerase chain reaction. First, we assessed the influence of MAFLD on distinct microbial communities at the bacterial phylum level. Next, the correlation between the microbial communities and diversity in patients with MAFLD was evaluated. RESULTS: Among the enrolled participants, the non-MAFLD and MAFLD groups consisted of 21 and 22 patients, respectively. Sequences were distributed among ten bacterial phyla. The relative abundance of Firmicutes was significantly higher in the MAFLD group than in the non-MAFLD group (p = 0.014). The microbial diversity was not significantly influenced by the presence of MAFLD (Chao-1 index: p = 0.215 and Shannon index: p = 0.174, respectively); nonetheless, the correlation coefficient between the abundances of Firmicutes and microbial diversity was higher in the non-MAFLD group than in the MAFLD group. CONCLUSION: The presence of MAFLD increased the relative abundances of Firmicutes at the bacterial phylum level, which may cause the discrepancy between the abundances of Firmicutes and diversity in patients with MAFLD.

Impact of liver fibrosis on the relative abundance of a urease-positive Streptococcus salivarius group from saliva in patients with chronic liver disease.

AIM: We performed genomic analysis to study the relative abundance of a urease-positive Streptococcus salivarius group isolated from the saliva of patients with chronic liver disease. METHODS: Male and female patients with chronic liver disease aged over 20 years were included. First, we assessed the frequency and type of the S. salivarius group isolated from oral saliva using molecular biology techniques based on 16S rRNA and dephospho-coenzyme A kinase gene sequencing. Next, we assessed the correlation between the urease positivity rate in the S. salivarius group isolated from oral saliva and liver fibrosis based on chronic liver disease. Urease-positive strains were identified by the urease test using urea broth (Difco, Franklin Lakes, NJ, USA). Liver fibrosis was evaluated by the liver stiffness measurement value based on magnetic resonance elastography. RESULTS: A total of 45 patients identified using the multiplex polymerase chain reaction for the 16S rRNA gene were tested using the multiplex polymerase chain reaction for the dephospho-coenzyme A kinase gene. Confirming the strains detected in each of the 45 patients, urease-positive S. salivarius was detected in 28 patients (62%), urease-negative S. salivarius in 25 patients (56%), and urease-positive Streptococcus vestibularis in 12 patients (27%). There was no patient with urease-negative S. vestibularis. The urease-positive rate of the S. salivarius group in the cirrhosis and non-cirrhosis groups were 82.2% and 39.2%, respectively. The liver cirrhosis group had a higher urease positivity rate than the non-cirrhotic group (p < 0.001). CONCLUSIONS: Liver fibrosis influences the frequency of a urease-positive S. salivarius group isolated from oral saliva.

Current Prevalence of Antimicrobial Resistance in Okayama from a National Database between 2018 and 2021.

Antimicrobial resistance is an emerging global threat that must be addressed using a multidisciplinary approach. This study aimed to raise awareness of high-level antimicrobial-resistant (AMR) pathogens in Japan by comparing their recent prevalences among prefectures, particularly Okayama. Data for the isolation proportions of meropenem-resistant Pseudomonas aeruginosa, methicillin-resistant Staphylococcus aureus, vancomycin-resistant Enterococcus faecium, cefotaxime-resistant Escherichia coli and Klebsiella pneumoniae, and levofloxacin-resistant E. coli and K. pneumoniae were collected from the Japan Nosocomial Infections Surveillance, a national database sponsored by the Japanese Ministry of Health, Labour, and Welfare, between 2018 and 2021. The average isolated proportions of the seven AMR pathogens were higher in Okayama compared to other prefectures: the worst (19.9%) was meropenem-resistant P. aeruginosa, the sixth worst (57.2%) was methicillin-resistant S. aureus, the eighth worst (3.3%) was vancomycin-resistant E. faecium, the second (37.8%) and fifth worst (17.6%) were cefotaxime-resistant E. coli and K. pneumoniae, respectively, and the fourth (49.9%) and third worst (8.7%) were levofloxacin-resistant E. coli and K. pneumoniae, respectively. Our study highlights the notably high prevalences of representative AMR pathogens in Okayama, suggesting the need for fundamental infection prevention and control by healthcare professionals, promoting antimicrobial stewardship, and educating undergraduates and postgraduates in Okayama.

Prevalence of Inducible Macrolide, Lincosamide, and Streptogramin B (inducible MLSB) Resistance in Clindamycin-Susceptible Staphylococcus aureus at Okayama University Hospital.

Inducible resistance to the macrolide, lincosamide, and streptogramin B (iMLSB) antibiotic family is a latent mechanism for antimicrobial resistance in Staphylococcus aureus. We here investigated the frequency and genotypic profiles of iMLSB resistance in clindamycin (CLDM)-susceptible S. aureus isolated in Okayama University Hospital from June 2020 to June 2021. We phenotypically screened the iMLSB resistance via D-zone test and performed PCR testing for the erythromycin ribosomal methylase (erm) genes: ermA and ermC. Among 432 CLDM-susceptible S. aureus isolates, 138 (31.9%) exhibited an iMLSB-resistance phenotype, with methicillinresistant S. aureus isolates (MRSA; 61 isolates: 58.6%) exhibiting higher positivity than methicillin-sensitive S. aureus isolates (MSSA; 77 isolates: 23.5%) (p<0.001). Male patients had a higher frequency of iMLSB resistance than females (OR [95%CI]: 1.8 [1.2-2.8]; p=0.007). Genotypically, ermA predominated in both MSSA (70.1%) and MRSA (86.9%) compared to ermC (14.3% in MSSA and 11.5% in MRSA). A single strain of MRSA possessed both ermA and ermC, while 12 (15.6%) MSSA isolates were negative for both ermA and ermC, suggesting the presence of other genetic mechanisms. Collectively, these results show that approximately 33% of CLDM-susceptible S. aureus isolates at our university hospital exhibited iMLSB resistance, predominantly caused by ermA in both MSSA and MRSA.

RNA Editing is a Valuable Biomarker for Predicting Carcinogenesis in Ulcerative Colitis.

BACKGROUND AND AIMS: Ulcerative colitis [UC] can lead to colitis-associated colorectal neoplasm [CAN]. Adenosine-to-inosine RNA editing, which is regulated by adenosine deaminase acting on RNA [ADAR], induces the post-transcriptional modification of critical oncogenes, including antizyme inhibitor 1 [AZIN1], leading to colorectal carcinogenesis. Therefore, we hypothesized that ADAR1 might be involved in the development of CAN in UC. METHODS: We systematically analysed a cohort of 139 UC cases [40 acute phase, 73 remission phase, 26 CAN]. The degree of inflammation was evaluated using the Mayo endoscopic score [MES]. RESULTS: The type 1 interferon [IFN]-related inflammation pathway was upregulated in the rectum of active UC, rectum of UC-CAN and tumour site of UC-CAN patients. ADAR1 expression was upregulated in the entire colon of CAN cases, while it was downregulated in non-CAN MES0 cases. ADAR1 expression in the rectum predicted the development of CAN better than p53 or ß-catenin, with an area under the curve of 0.93. The high expression of ADAR1 and high AZIN1 RNA editing in UC was triggered by type 1 IFN stimulation from UC-specific microbiomes, such as seen in Fusobacterium in vitro analyses. The induction of AZIN1 RNA editing by ADAR1, whose expression is promoted by Fusobacterium, may induce carcinogenesis in UC. CONCLUSIONS: The risk of CAN can be evaluated by assessing ADAR1 expression in the rectum of MES0 UC patients, freeing UC patients from unnecessary colonoscopy and reducing their physical burden. RNA editing may be involved in UC carcinogenesis, and may be used to facilitate the prevention and treatment of CAN in UC.

Effects of Helicobacter pylori and Nitrate-Reducing Bacteria Coculture on Cells.

Helicobacter pylori infection is an important risk factor for developing gastric cancer. However, only a few H. pylori-infected people develop gastric cancer. Thus, other risk factors aside from H. pylori infection may be involved in gastric cancer development. This study aimed to investigate whether the nitrate-reducing bacteria isolated from patients with atrophic gastritis caused by H. pylori infection are risk factors for developing atrophic gastritis and gastric neoplasia. Nitrate-reducing bacteria were isolated from patients with atrophic gastritis caused by H. pylori infection. Among the isolated bacteria, Actinomyces oris, Actinomyces odontolyticus, Rothia dentocariosa, and Rothia mucilaginosa were used in the subsequent experiments. Cytokine inducibility was evaluated in monocytic cells, and mitogen-activated protein kinase (MAPK) activity and cell cycle were assessed in the gastric epithelial cells. The cytotoxicities and neutrophil-inducing abilities of the Actinomyces and Rothia species were enhanced when cocultured with H. pylori. Th1/Th2-related cytokines were also expressed, but their expression levels differed depending on the bacterial species. Moreover, H. pylori and Actinomyces activated MAPK (ERK and p38) and affected cell cycle progression. Some nitrate-reducing bacteria cocultured with H. pylori may promote inflammation and atrophy by inducing cytokine production. In addition, the MAPK activation and cell cycle progression caused by these bacteria can contribute to gastric cancer development.

Detection of Enterobacter cloacae complex strain with a blaNDM-1-harboring plasmid from an elderly resident at a long-term care facility in Okayama, Japan.

Amidst the global spread of antimicrobial resistance, New Delhi metallo-ß-lactamase (NDM)-type carbapenemase-producing Enterobacterales (CPE) remain uncommon in Japan, and the detection of such highly drug-resistant organisms is limited to inbound cases. There is little evidence regarding the prevalence of NDM ß-lactamase gene (blaNDM)-harboring CPE in the domestic community, especially in the provincial cities of Japan. Herein, we report the isolation of a blaNDM-1-harboring plasmid in Enterobacter cloacae complex strain isolated from an elderly woman without a history of traveling abroad who had resided in a long-term care facility in Okayama, Japan. The multidrug-resistant blaNDM-harboring CPE isolate was detected in a stool sample of the patient during routine screening at admission. We performed whole-genome sequencing analysis of the isolate using MiSeq (Illumina) and MinION (Oxford Nanopore Technologies) platforms. The isolate was identified as sequence type 171, which has predominantly been reported in the United States and China. The blaNDM-1 gene was encoded on the 46,161 bp IncX3 plasmid, with sequence similarity to plasmids of similar size isolated from individuals in China. Collectively, the genomic data suggest that an imported CPE isolate may have spread among healthy individuals in the regional area of Japan.

Phylogenic analysis of new viral cluster of large phages with unusual DNA genomes containing uracil in place of thymine in gene-sharing network, using phages S6 and PBS1 and relevant uncultured phages derived from sewage metagenomics.

Bacteriophages (phages) are the most diverse and abundant life-form on Earth. Jumbophages are phages with double-stranded DNA genomes longer than 200 kbp. Among these, some jumbophages with uracil in place of thymine as a nucleic acid base, which we have tentatively termed "dU jumbophages" in this study, have been reported. Because the dU jumbophages are considered to be a living fossil from the RNA world, the evolutionary traits of dU jumbophages are of interest. In this study, we examined the phylogeny of dU jumbophages. First, tBLASTx analysis of newly sequenced dU jumbophages such as Bacillus phage PBS1 and previously isolated Staphylococcus phage S6 showed similarity to the other dU jumbophages. Second, we detected the two partial genome sequences of uncultured phages possibly relevant to dU jumbophages, scaffold_002 and scaffold_007, from wastewater metagenomics. Third, according to the gene-sharing network analysis, the dU jumbophages, including phages PBS1 and S6, and uncultured phage scaffold_002 formed a cluster, which suggested a new viral subfamily/family. Finally, analyses of the phylogenetic relationship with other phages showed that the dU jumbophage cluster, which had two clades of phages infecting Gram-negative and Gram-positive bacteria, diverged from the single ancestral phage. These findings together with previous reports may imply that dU jumbophages evolved from the same origin before divergence of Gram-negative and Gram-positive bacteria.

Detection of in-frame mutation by IS30-family insertion sequence in the phospholipid phosphatidylglycerol synthase gene (pgsA2) of high-level daptomycin-resistant Corynebacterium striatum.

The emergence of high-level daptomycin (DAP)-resistant (HLDR) Corynebacterium striatum has been reported as a result of loss-of-function point mutations or premature stop codon mutations in a responsible gene, pgsA2. We herein describe the novel detection of an HLDR C. striatum clinical isolate, in which IS30-insertion was corroborated to cause destruction of pgsA2 gene. We isolated an HLDR C. striatum from a critically ill patient with underlying mycosis fungoides who had been treated with DAP for 10 days. With a sequence investigation, IS30-insertion was discovered to split pgsA2 in the HLDR C. striatum strain, which may cause disrupted phospholipid phosphatidylglycerol (PG) production. Future studies should survey the prevalence of IS-mediated gene inactivation among HLDR C. striatum clinical isolates.

Fecal microbiota transplantation as therapy for recurrent Clostridioides difficile infection is associated with amelioration of delirium and accompanied by changes in fecal microbiota and the metabolome.

Recurrent Clostridioides difficile infection (rCDI) is a frustrating condition that may affect a person's quality of life for months. Microbiome-based therapy such as fecal microbiota transplantation (FMT) has been effective for the treatment of rCDI by correcting the imbalance of the gut microbiota. Appropriate antibiotic treatment is recommended for at least two recurrences before offering FMT. Here, we report the case of a 92-year-old woman who experienced five recurrences of Clostridioides difficile infection (CDI) (six episodes in total) complicated by dementia and delirium, both of which were dramatically improved by FMT, which was associated with alterations in fecal microbiota and the metabolome. Analyses of whole microbial communities and metabolomic analyses were performed on stool specimens collected from the patient on the first episode, the third episode, the day of FMT (before FMT), and 2, 8, and 23 weeks after the FMT and from the donor. The patient had various fecal dysbioses on the first and third episodes and on the day of FMT. Two weeks after FMT, diversity of the gut bacteriome as well as the virome increased dramatically and was reflected in a positive clinical outcome for this patient. Metabolomic analysis revealed that short-chain fatty acids, which have been reported to be associated with improved memory function, were increased after FMT.

In vitro effectiveness of biapenem against IMP-producing Enterobacteriaceae.

The options available for treating infections with carbapenemase-producing Enterobacteriaceae (CPE) are limited; with the increasing threat of these infections, new treatments are urgently needed. Biapenem (BIPM) is a carbapenem, and limited data confirming its in vitro killing effect against CPE are available. In this study, we examined the minimum inhibitory concentrations (MICs) and minimum bactericidal concentrations (MBCs) of BIPM for 14 IMP-1-producing Enterobacteriaceae strains isolated from the Okayama region in Japan. The MICs against almost all the isolates were lower than 0.5 µg ml-1, indicating susceptibility to BIPM, while approximately half of the isolates were confirmed to be bacteriostatic to BIPM. However, initial killing to a 99.9 % reduction was observed in seven out of eight strains in a time-kill assay. Despite the small data set, we concluded that the in vitro efficacy of BIPM suggests that the drug could be a new therapeutic option against infection with IMP-producing CPE.

Serodiagnosis and Bacterial Genome of Helicobacter pylori Infection.

The infection caused by Helicobacter pylori is associated with several diseases, including gastric cancer. Several methods for the diagnosis of H. pylori infection exist, including endoscopy, the urea breath test, and the fecal antigen test, which is the serum antibody titer test that is often used since it is a simple and highly sensitive test. In this context, this study aims to find the association between different antibody reactivities and the organization of bacterial genomes. Next-generation sequences were performed to determine the genome sequences of four strains of antigens with different reactivity. The search was performed on the common genes, with the homology analysis conducted using a genome ring and dot plot analysis. The two antigens of the highly reactive strains showed a high gene homology, and Western blots for CagA and VacA also showed high expression levels of proteins. In the poorly responsive antigen strains, it was found that the inversion occurred around the vacA gene in the genome. The structure of bacterial genomes might contribute to the poor reactivity exhibited by the antibodies of patients. In the future, an accurate serodiagnosis could be performed by using a strain with few gene mutations of the antigen used for the antibody titer test of H. pylori.

Antibacterial Effects of Disulfiram in Helicobacter pylori.

BACKGROUND: Helicobacter pylori infection poses a risk of the occurrence of gastrointestinal diseases, such as gastric cancer. Its incidence rate is significantly reduced by eradication, and thereby, eradication therapy is generally performed. Disulfiram is an oral prescription drug mainly used for the treatment of alcohol dependence. In recent years, reports have been made on its anticancer and antibacterial effects, and thus, it has recently become an interesting subject. This study aimed to examine the antibacterial activity of disulfiram, investigate the presence or absence of its antibacterial activity on H. pylori, and determine whether it could be a new bactericidal drug against drug-resistant H. pylori. MATERIALS AND METHODS: Drug-sensitive strains of H. pylori and amoxicillin-resistant, clarithromycin-resistant, and metronidazole-resistant strains were used, and a growth inhibition test of H. pylori using disulfiram was performed. Furthermore, the expression of urease, vacuolating cytotoxin A (VacA), and CagA, the virulence proteins of H. pylori, was quantitatively analyzed using the Western blotting method. In addition, for H. pylori used in this study, the 16SrDNA sequence, a ribosomal gene involved in protein production, was analyzed to examine the presence or absence of gene mutation. RESULTS: Disulfiram suppressed the growth of 7 out of 12 H. pylori strains at 1 µg/mL, and no correlation was observed between their susceptibility/resistance to current eradication antimicrobial drugs and disulfiram resistance. Disulfiram reduced the expression levels of urease, VacA, and CagA proteins. H. pylori, which showed resistance to disulfiram, tended to have fewer gene deletions/insertions in the 16S rDNA sequence; however, no specific mutation was detected. CONCLUSION: Disulfiram has a bactericidal effect on H. pylori at low concentrations, suggesting that it can be used as a supplement for current H. pylori eradication drugs.

Elizabethkingia anophelis, an emerging pathogen, inhibits RAW 264.7 macrophage function.

Elizabethkingia anophelis is a pathogen that can cause a life-threatening infection in immunocompromised patients. The first case of E. anophelis infection was reported in 2013; subsequently, an increase in its incidence has been reported globally. Additionally, a mortality rate of more than 30% was observed in the US outbreak of 2015. To date, the pathogenic mechanisms underlying E. anophelis infection, such as toxin production, remain unclear. Since tissue macrophages act as the first line of defense against pathogens, in the present study the interactions between E. anophelis and a macrophage-like cell line RAW 264.7 were examined. Although E. anophelis showed no cytotoxicity toward RAW 264.7 macrophages, the infection inhibited LPS-induced morphological changes and activation of differentiation markers for the polarization of RAW 264.7 macrophages toward an M1-like phenotype. However, when the cell contact was restricted using Transwell inserts or bacterial culture supernatants were used instead of live bacteria, no such inhibition was observed. Moreover, it was shown that E. anophelis evaded phagocytosis. Overall, the results suggest that E. anophelis infection inhibits the differentiation of RAW 264.7 macrophages to a pro-inflammatory phenotype in a contact-dependent manner.

Septic pulmonary emboli caused by Tsukamurella inchonensis: A case report.

The genus Tsukamurella is a fastidious, environmental organism that potentially causes various infections in humans. Due to the morphological and biochemical similarities to others pathogens, such as Gordona, Rhodococcus, Corynebacterium, Nocardia, and Mycobacterium, a molecular-based approach is indispensable to correctly identify them. Herein, we describe a case of Tsukamurella inchonensis bacteremia complicated with septic pulmonary emboli (SPE), which is the first in the literature. A 44-year-old Japanese woman diagnosed with tongue cancer had undergone partial tongue resection. While receiving chemotherapy and radiotherapy, she developed high fever. Chest computed tomography suggested multiple emboli at the bilateral, peripheral lungs, indicating SPE. Blood culture detected Gram-positive rods, to which matrix-assisted laser desorption/ionization-time of flight mass spectrometry failed to identify. Then, we attempted to characterize it by 16S rRNA sequence, which suggested the organism to be Tsukamurella species but resulted in low resonance of the species-level identification. Additionally, we employed a confidence gene targeting groEL, leading to 100% matching (753/753 bps) with T. inchonensis NCTC 10741 (GenBank accession no. LR131273.1), which has been incorrectly registered as wrong species name Tsukamurella paurometabola in the database. Under the diagnosis of T. inchonensis-associated SPE, we successfully treated the patient with imipenem/cilastatin administration for 4 weeks. Sequencing analysis of groEL was of great use in identifying the organism in this case. More clinical cases based on molecular diagnosis of the fastidious pathogens need to be accumulated to further understand the characteristics and appropriate treatment regimen.

Enhancement of intestinal epithelial barrier function by Weissella confusa F213 and Lactobacillus rhamnosus FBB81 probiotic candidates in an in vitro model of hydrogen peroxide-induced inflammatory bowel disease.

OBJECTIVE: Weissella confusa F213 (WCF213) and Lactobacillus rhamnosus FBB81 (LrFBB81) are two probiotic candidates isolated from humans in our previous study. Their functional activity on the mucosal barrier has not yet been adequately investigated. Therefore, the objective of this study was to investigate the effect of these strains on maintaining mucosal integrity in vitro. Caco-2 cell monolayers were pretreated with WCF213 and LrFBB81 before being exposed to hydrogen peroxide. The integrity of mucosal cells was evaluated by measuring the transepithelial resistance (TER), flux of FITC-labelled dextran, and ZO-1 protein distribution with the help of an immunofluorescence method. RESULTS: WCF213 was found to significantly maintain the TER better than the control hydrogen peroxide-treated cells (p < 0.001), followed by the strain combination, and LrFBB81 alone (p < 0.05). The permeability of mucosa was also successfully maintained by the WCF213 strain. This was illustrated by the significant reduction in the flux of FITC-labelled dextran (p < 0.05), which was larger than that exhibited by the other groups. The ZO-1 distribution of strain-treated cells showed less disruption than hydrogen peroxide-treated cells, consistent with the TER and FITC experimental results. These findings indicate that WCF213 and LrFBB81 plays important roles in the maintenance of mucosal integrity in a strain-dependent manner.

Analysis of a plasmid encoding botulinum neurotoxin type G gene in Clostridium argentinense.

Clostridium argentinense produces botulinum neurotoxin type G (BoNT/G). We sequenced and analyzed the plasmid harboring the bont/G gene, designated pCAG, in C. argentinense strain 2740. The pCAG consisted of 140,070 bp containing the bont/G gene cluster. Although this gene cluster showed high similarities in its DNA sequence and ORF arrangement to those of other bont gene clusters, the other regions of the plasmid did not. A phylogenetic study suggested that pCAG had a unique evolutionary history compared with other clostridial bont-harboring plasmids. This suggests that pCAG is possibly a novel type of plasmid expressing the bont/G gene in C. argentinense.

Export of a Vibrio parahaemolyticus toxin by the Sec and type III secretion machineries in tandem.

Many Gram-negative pathogens utilize dedicated secretion systems to export virulence factors such as exotoxins and effectors1-4. Several exotoxins are synthesized as precursors containing amino-terminal Sec signal peptides and are exported through the inner-membrane-bound Sec machinery to the periplasm, followed by secretion across the outer membrane to the exterior using a type II secretion system (T2SS)3,5. Here, we report that thermostable direct haemolysin (TDH), an exotoxin of the food-borne pathogen Vibrio parahaemolyticus, can be exported through the type III secretion system (T3SS), which engages in one-step secretion of effectors4, despite possessing a Sec signal peptide and being mainly secreted via the T2SS. Although the precursor of TDH is targeted to the Sec pathway, a fraction of mature TDH was observed to re-enter the bacterial cytoplasm. The N terminus of mature TDH comprises a T3SS signal sequence, allowing it to be loaded into the T3SS. We also show that T3SS-delivered TDH as an effector contributes to intestinal fluid accumulation in a rabbit diarrhoeal model of V. parahaemolyticus infection. Thus, our results show that an unconventional export mechanism for a bacterial toxin via the T3SS in tandem with the Sec machinery facilitates the virulence trait of V. parahaemolyticus.

Vibrio alginolyticus VepA Induces Lysosomal Membrane Permeability and Cathepsin-Independent Cell Death.

The bacterium Vibrio alginolyticus, an opportunistic pathogen in humans, has a type III secretion system (T3SS) that is responsible for its cytotoxicity toward eukaryotic cells. The effector of T3SS that is responsible for the cytotoxicity had not been identified. Here we demonstrate that VepA, a homolog of the T3SS effector in V. parahaemolyticus, is required for cytotoxicity in V. alginolyticus. VepA induces lysosomal membrane permeabilization, and it allows the leakage of only small molecules into the cytosol. Our findings revealed that VepA induces cathepsin-independent cell death in mammalian cells. The ferrous ion, one of the small molecules in the lysosome contents, appears to be involved in the cell death caused by V. alginolyticus VepA.